Human breast carcinoma cell line capable of production of a spontaneously metastasizing tumor in animals for use in anticancer drug testing

ABSTRACT

An adherent, stable, continuous human breast carcinoma cell line (GI-101A) has been produced from an infiltrating ductal breast carcinoma xenograft (GI-101) which has been grown and maintained in athymic mice for the past nine years. The GI-101A cells grow with an average doubling time of about 48 to about 72 hours. The cells display antigenic determinants consistent with those of the human breast tumor xenograft (GI-101) from which it was derived. The cell line, GI-101A, when injected subcutaneously into the subaxial area of athymic animals, such as athymic mice, produces tumors that spontaneously metastasize to distant organ sites, such as the lungs and lymph nodes. The cell line and the tumors that it produces may be used as model systems for study mechanisms responsible for metastatic behavior and for testing for new as well as screening for effective new anti-cancer drug therapies.

This is a division of application Ser. No. 08/350,938, filed Dec. 7,1997 U.S. Pat. No. 5,693,533.

FIELD OF THE INVENTION

This invention relates to the fields of tumor cell biology, metastasisresearch, immunochemistry, molecular biology, and anticancer drugdevelopment and screening. More particularly, the present invention isconcerned with a stable metastatic human breast carcinoma cell line thatis suitable for in vitro and in vivo anticancer drug development andscreening.

BACKGROUND

The 1992 cancer statistics estimated that 181,000 new cases of invasivebreast would be diagnosed that year and result in 46,300 new deaths. SeeBoring, C. C. et al.; Cancer, 42:19 (1992). In North America, breastcancer is the most common malignancy of women and accounts for 27% ofall female cancers and 18% of all female cancer mortalities. For womenaged 40-55, breast cancer represent the leading cause of death overall.While the age standardized incidence of breast cancer has risen annually(1-4% per year), the age adjusted mortality has remained stable for thepast fifty years (in the U.S.A.). Most women that die from breast cancersuccumb not to the original primary disease, which is usually amenableto various therapies, but rather from metastatic spread of the breastcancer to distant sites. This fact underscores the need to developeither novel anticancer agents or more aggressive forms of therapydirected specifically against the metastatic breast tumor cell.Requisite to the development of new treatment modalities is afundamental, thorough understanding of the regulatory processes inherentto the growth of both the primary and metastatic breast cancer cell andtumor. This process has been severely hampered by the lack ofappropriate and clinically relevant modeling systems. The modeldescribed here can be useful in providing the latter.

Anticancer drug screening trials have been conducted for many yearsusing a wide variety of tumor cell lines as targets. Drugs that showanticancer activity in vitro are subsequently tested against an in vivotumor model. While there are, in all, a large number of human tumor celllines, the availability of human breast cancer cell lines is limited. Ofthe few human breast tumor cell lines that are even capable of producinga tumor in immune suppressed animals, fewer still are capable ofproducing a metastasizing tumor in the animal except under extreme formsof manipulation. Cell lines like MDA-MB-231 and MDA-MB-435, see Price,J. E. et al.; Cancer Res., 50:717-721 (1990), produce consistentlymetastasizing tumors when the cells are injected directly into themammary fat pad of animals, such as athymic mice. On the other hand,subcutaneous injection of these cells produce tumors that are lessconsistently metastatic and have a different metastatic pattern thantumors implanted directly to the mammary fat pad. What is needed arecells that can produce a metastasizing tumor without the need forsurgical or experimental manipulations. The invention (GI-101A)described here, as well as the xenograft line from which it was derived,consistently produce tumors and lung metastases from subcutaneousimplants, thus requiring no additional experimental manipulations.

The Goodwin Institute for Cancer Research has previously developed andreported a spontaneously metastasizing human breast tumor xenograftmodel (GI-101). See Hurst, J. et al.; Cancer, 68:274-276 (1993). Thissolid tumor model, maintained through serial animal transplantation, hasbeen described in detail. In brief, the tumor presents as a poorlydifferentiated mammary carcinoma with occasional acinar and ductalformation. The tumor xenograft when implanted subcutaneously growsslowly and eventually metastasizes to the lungs, lymph nodes and bonemarrow of athymic murine recipients. This invention is the developmentof a cell line (GI-101A) from the GI-101 xenograft that is suitable forboth in vitro and in vivo investigations.

SUMMARY OF THE INVENTION

The prevent invention overcomes certain of the above-mentionedshortcomings and drawbacks and is directed to a novel, stable,continuous, human aneuploid breast carcinoma cell line which has theability to produce metastatic solid tumors at distant organ sites, suchas the lungs and lymph nodes, when introduced into athymic nude animals,like athymic nude mice. Surprisingly, the cell line of the presentinvention is able to consistently produce solid tumors and lungmetastases from subcutaneous and/or injected implants without requiringadditional experimental manipulations. Moreover, the cell line of thepresent invention constitutes a unique system that is suitable fordetecting and screening for new and effective anti-cancer therapies.This may be accomplished by, for instance, introducing into an athymicnude animal having the cell line of the present invention introducedtherein, a drug in an effective amount and analyzing the animal todetermine the effectiveness of the drug against one or more or all ofthe solid tumor metastases produced in the animal by the cell line atthe distant organs.

While the present invention is described herein with reference to theparticular cell line disclosed, it should nevertheless be understoodthat those skilled in this art that the present invention contemplatesequivalents to the cell line which have identical or substantiallysimilar characteristics and activity. Therefore, the present inventionincludes any cell line which has the ability to accomplish theobjectives of the instant invention, i.e., any human aneuploid breastcarcinoma cell line having a panel of human and tumor markers asidentified in Table I below which, when implanted or injected into anathymic nude animal, produces solid tumor metastases at distant organsites, such as the lungs and the lymph nodes, and may be used as in vivoand in vitro model systems to detect and screen for new and effectiveanti-cancer therapies.

The above features and advantages of the present invention will bebetter understood with reference to the accompanying DetailedDescription. It should also be understood that the particular methodsand cell lines illustrating the invention are exemplary only and are notto be regarded as limitations of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The cell line is derived from a human breast tumor xenograft line(GI-101) which originates as a recurrent infiltrating ductal mammaryadenocarcinoma of a 57 year old female (Stage IIIa, T3N2MX). Thisxenograft line is poorly differentiated and presents with occasionalductal and acinar formation. The tumor line is grown and maintainedthrough serial animal transplantation in athymic mice for over nineyears.

Tumor xenograft material is asceptically harvested from an animalbearing the GI-101 breast tumor xenograft [transplant generation #21;tumor volume>2000 mm³ ]. The xenograft is then dissected of connectiveand fatty tissue and gross necrotic areas and the remaining tissuemechanically dissociated through a 200 mesh sterile stainless steel cellsieve (Sigma) into RPMI 1640 tissue culture medium (Sigma) containing10% fetal calf serum (Flow) and supplemented with 200 mM glutamine and50 ug/ml of gentamycin (Sigma). The tumor brie is washed three times inphosphate-buffered saline at 150×g to remove dead cells. Viable cellcounts are made and the cells seeded (1×10⁶ cells/ml) in 5 ml of tissueculture-medium, as described above, in 25 cm² tissue culture flasks andincubated at 37° C., 5% CO₂ in air and 99% relative humidity. Thecultures are demi-fed weekly with fresh media and recharged bi-weeklyfor eight weeks with fresh tumor cells using the same procedure outlinedabove for obtaining the original tumor brie. The cultures are maintainedas an adherent cell line, removing non-adherent cells for eachsubculture. Subcultures are performed by detaching adherent cells with0.05% trypsin-EDTA (Sigma). As soon as cells are detached, cellviability in number are counted and the cells seeded as described above.Fibroblasts disappear after three months of continuous culture followingcessation of the last tumor cell recharge. Upon fresh culture, the cells(GI-101A) grow with a lag phase of about 48 to about 72 hours followedby a doubling time of approximately sixty hours. In culture, the cellsgrow as sheets of epithelial-like cells, which may pile up intospherical colonies after the culture reaches confluency.

The tumorigenicity of the GI-101A cells is originally confirmed byinjecting 60×10⁶ cells from in vitro passage #6 into the subcutaneoussubaxillary area of five 10 week-old female athymic nude mice. Beforeinjection, the cells are treated with EDTA (200 ug/ml) to detach themfrom the tissue culture flask. The cells are then washed three times bycentrifugation at 400×g and resuspended in serum-free RPMI 1640 tissueculture medium for injection. The histology of the primary tumorsproduced from injection of GI-101A cells result in moderate to welldifferentiated tumors showing lymphocyte infiltration and some areas ofnecrosis. Serial transplantation of these tumors results in tumors withthe same growth characteristics as the original GI-101 xenograft anddisplay a progressive loss of differentiation as the proportion ofmurine stromal elements increases with each transplant generation. Bythe third serial transplantation, the tumors derived resemble theoriginal GI-101 xenograft. The tumors produced by injection of GI-101Atumor cells and subsequent serial transplantation spontaneouslymetastasize distant organ sites, such as the lungs and lymph nodes.

The cell line is believed to be human and aneuploid by histogramanalysis. Two major cell populations are apparent.

In addition, the cell line is characterized for a panel of human andtumor markers, as reported in Table I by the methods listed below.

                  TABLE I                                                         ______________________________________                                                                  Reactivity                                                                             Method of                                    Marker Description * Analysis**                                             ______________________________________                                          MC5 Breast Tumor Antigen +++ F,H                                              CEA Carcinoembryonic Antigen +++ F,H                                          CK Cytokeratin (human) ++++ F,H                                               PCNA Proliferating Cell Nuclear Antigen ++ F,H                                P120 Proliferation Antigen +++ F,H                                            P105 Proliferation Antigen +++ F                                              EGF-r Epidermal Growth Factor Receptor +++ F,H                                EMA Epithelial Membrane Antigen ++ F,H                                        KC4 Cytokeratin (human) +++ H                                                 LAM L-Selectin Adhesion Molecule +/-- F                                       VCAM Vascular Cell Adhesion Molecule -- F                                     ELAM Endothelial Leukocyte Adhesion -- F                                       Molecule                                                                     ICAM Intercellular Adhesion Molecule + F                                      IL-2r(p75) Interleukin 2 Receptor -- F                                         (mw=75 kD)                                                                   IL-2r(p55) Interleukin 2 Receptor -- F                                         (mw=55 kD)                                                                   CD15 Neutrophil Marker ++ F                                                   CD45 Monocyte Marker -- F                                                     CD38 Activated Lymphocyte/Basophil/ -- F                                       Monocyte Marker --                                                           CD34 Immature Granoluocyte Marker -- F                                        APO-1 Apoptosis 1 Marker -- F                                                 FEL Leukemia associated oncogene + P                                          NM23 Metastasis Associated Marker -- P                                        MDM2 Suppressor of p53 oncogene ++ H,W                                        TGF-a Transforming Growth Factor alpha ++ E,H                               ______________________________________                                        *Reactivity      *Method of Analysis                                          ______________________________________                                        --    =      None        E   =    ELISA                                         + = Low - Moderate F = Flow Cytometry                                         ++ = Moderate H = Immunocytochemistry                                         +++ = Moderate - High P = Polymerase Chain                                         Reaction                                                                 ++++ = High W = Western Blot                                                ______________________________________                                    

Commercial kits as prepared by Oncogene Science are used to determinethe levels and expression of TGF-a.

Cytospin slides prepared using the GI-101A cells are prepared andstained with biotinylated primary monoclonal antibody (i.e., EGF, etc.),followed by streptavidin conjugated peroxidase with ABTS/H₂ O₂(2,2'Azino-di-(3-Ethylbenzthiozoline Sulfonic acid) as a substratechromogen. The slides are counter stained with hematoxylin and eosin.

Flow cytometric analysis for proliferation associated nuclear or cellsurface antigens are performed by the methods reported in Bolton, et al.See Bolton, W. E. et al.; Cytometry, 13(2):117-126 (1992). Briefly,whole cells or nuclei (prepared by Coulter DNA-Prep) are incubated with10 ug of specific antibody (PCNA, etc.). Following lysis and propidiumiodide staining (for nuclei) or washing, the cells are analyzed using anupgraded Profile I Flow Cytometer (Coulter Corp., Hialeah, Fla.) fittedwith an argon laser and equipped with an EPICS ELITE workstation usingthe Multicycle software analysis package developed by P. S. Rabinovitch(Phoenix Flow Systems, San Diego, Calif.). DNA histograms for all cellpreparations are constructed along with data analysis for percentage ofpositive cells and stain intensity for those cells.

The gene expression for Fel is analyzed using PCR primers identifyingprevious work. Total RNA is isolated, first strand cDNAs synthesizedusing BRL/Gibco Superscript reverse transcriptase, and target sequencesamplified with Taq DNA polymerase under manufacturers' directions.Strict quantitative analysis is first performed on human β-actin andtransferrin receptor expression by constructing competitors as follows:Amplification of non-relevant sequence (FEL cDNA from B-ALL) withprimers containing extended regions of β-actin or transferrin receptorhomology. Those competitor constructs are cloned into a bluescriptvector (SK-) containing a 3'A.sub.(20) cassette. The competitor plasmidsare then linearized and "mRNA" transcribed with a Promega Ribomaxtranscription kit and T7 RNA polymerase, and isolated using anInvitrogen micro-fast-tract poly A isolation kit. Poly A RNA is thenspectrophotometrically quantitated, and known amounts added to totaltumor RNA samples. These samples are then amplified with β-actin andtransferrin receptor sequences, and both endogenous and competitor PCRproducts (300-500 bp) will be quantitated visually on ethidium stained3% nusieve agarose TAE gels. Subsequent semi-quantitative analysis onmetastasis associated gene expression will standardize against theseinternal controls by including β-actin and/or transferrin receptorprimers in each amplification reaction.

MDM2 expression is detected by SDS poly-acrylimide gel electrophoresisof the GI-101A cells. Electrophoresis is performed using a Bio-RadProtein II electrophoresis system. After electrophoresis, the proteinsare transferred to nitrocellulose membranes using a Bio-Rad Trans Blotcell. The membranes are blocked to reduce non-specific protein bindingand MDM2 detected using a specific anti-MDM2 monoclonal antibody. Thelabeled bands on the nitrocellulose membranes are visualized bytreatment with horse radish peroxidase conjugated secondary antibody anddetected by enhanced chemiluminescence techniques (Amersham, Rockford,Ill).

Cultures containing the subject GI-101A cell line have been deposited inthe permanent collection of the American Type Culture Collection (ATCC),12301 Parklawn Drive, Rockville, Md. 20852 USA on Oct. 22, 1997. Theaccession number for GI-101A is ATCC CRL-12420.

The subject culture has been deposited under conditions that assure thataccess to the culture will be available during the pendency of thispatent application to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 CFR 1.14 and 35 USC 122. Thedeposit is available as required by foreign patent laws in countrieswherein counterparts of the subject application, or its progeny, arefiled. However, it should be understood that the availability of adeposit does not constitute a license to practice the subject inventionin derogation of patent rights granted by governmental action.

Further, the subject culture deposit will be stored and made availableto the public in accord with the provisions of the Budapest Treaty forthe Deposit of Microorganisms, i.e., it will be stored with all the carenecessary to keep it viable and uncontaminated for a period of at leastfive years after the most recent request for the furnishing of a sampleof the deposit, and in any case, for a period of at least 30 (thirty)years after the date of deposit or for the enforceable life of anypatent which may issue disclosing the culture. The depositoracknowledges the duty to replace the deposit should the depository beunable to furnish a sample when requested, due to the condition of thedeposit. All restrictions on the availability to the public of thesubject culture deposit will be irrevocably removed upon the granting ofa patent disclosing it.

While the invention has been described with reference to a preferredembodiment, it should be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted forelements thereof without departing from the scope of the invention. Forinstance, the cell line or equivalents thereto may be used as modelsystems for study mechanisms responsible for metastatic behavior and fortesting and screening anti-cancer drug therapies in other suitableanimals, such as canines, bovines, rabbits, rats, pigs, sheep and thelike. In addition, many modifications may be made to adapt a particularsituation or material to the teachings of the invention withoutdeparting from the essential scope thereof. Therefore, it is intendedthat the invention not be limited to the particular embodiment disclosedas the best mode contemplated for carrying out the invention, but thatthe invention will include all embodiments falling within the scope ofthe appended claims.

Having described our invention, we claim:
 1. A solid tumor produced inan immune deficient non-human mammal having T-cell immunosuppressionwherein said tumor is spontaneously metastatic to distant organ sitesincluding lung and lymph node, wherein said tumor is produced byintroducing into said immune deficient mammal a human aneuploid breastcarcinoma cell line, wherein said cell line has the following markerprofile:a) positive for breast tumor antigen (MC-5), carcinoembyonicantigen (CEA), proliferating cell nuclear antigen (pCNA), proliferationantigens (p120 and p105), epidermal growth factor receptor (425 and528), and human cytokeratins (KC4), epithelial membrane antigen (EMA),neutrophil marker (CD 15), p53 oncogene suppressor protein (MDM2),transforming growth factor alpha (TGF-a), L-selectin adhesion molecule(LAM), intercellular adhesion molecule (ICAM), and leukemia associatedoncogene (FEL); and b) by negative for vascular cell adhesion molecule(VCAM), endothelial leukocyte adhesion molecule (ELAM), interleukin 2receptors (IL-2p75 and IL-2p55), monocyte marker (CD45), activatedlymphocyte/basophil/monocytemarker (CD38), immature granulocyte marker(CD34), apoptosis 1 marker (APO-1), and non-metastasis associated gene(NM23).
 2. An immune deficient non-human mammal having T-cellimmunosuppression and comprising one or more cells of a human aneuploidbreast carcinoma cell line, wherein said cell line has the followingmarker profile:a) positive for breast tumor antigen (MC-5),carcinoembyonic antigen (CEA), proliferating cell nuclear antigen(pCNA), proliferation antigens (p120 and p105), epidermal growth factorreceptor (425 and 528), and human cytokeratins (KC4), epithelialmembrane antigen (EMA), neutrophil marker (CD 15), p53 oncogenesuppressor protein (MDM2), transforming growth factor alpha (TGF-a),L-selectin adhesion molecule (LAM), intercellular adhesion molecule(ICAM), and leukemia associated oncogene (FEL); and b) negative forvascular cell adhesion molecule (VCAM), endothelial leukocyte adhesionmolecule (ELAM), interleukin 2 receptors (IL-2p75 and IL-2p55), monocytemarker (CD45), activated lymphocyte/basophil/monocyte marker (CD38),immature granulocyte marker (CD34), apoptosis 1 marker (APO-1), andnon-metastasis associated gene (NM23).
 3. The immune deficient mammal ofclaim 2, said immune deficient mammal being a mouse.
 4. The immunedeficient mammal of claim 2, said immune deficient mammal being selectedfrom the group consisting of canine, bovine, pig, rat, rabbit and sheep.5. A method of producing a solid tumor in an immune deficient non-humanmammal having T-cell immunosuppression wherein said tumor isspontaneously metastatic to distant organ sites including lung and lymphnode comprising:(a) obtaining a human aneuploid breast carcinoma cellline having the following marker profile:(1) positive for breast tumorantigen (MC-5), carcinoembyonic antigen (CEA), proliferating cellnuclear antigen (pCNA), proliferation antigens (p120 and p105),epidermal growth factor receptor (425 and 528), and human cytokeratins(KC4), epithelial membrane antigen (EMA), neutrophil marker (CD 15), p53oncogene suppressor protein (MDM2), transforming growth factor alpha(TGF-a), L-selectin adhesion molecule (LAM), intercellular adhesionmolecule (ICAM), and leukemia associated oncogene (FEL); (2) negativefor vascular cell adhesion molecule (VCAM), endothelial leukocyteadhesion molecule (ELAM), interleukin 2 receptors (IL-2p75 and IL-2p55),monocyte marker (CD45), activated lymphocyte/basophil/monocyte marker(CD38), immature granulocyte marker (CD34), apoptosis 1 marker (APO-1).and non-metastasis associated gene (NM23); and (b) introducing said cellline into said immune deficient non-human mammal, wherein said cell lineproduced a solid tumor in said mammal.